RXR homodimer formation

ABSTRACT

The invention provides a method of screening a substance for the ability to affect the formation of a retinoid X receptor homodimer comprising combining the substance and a solution containing retinoid X receptors and determining the presence of homodimer formation. Also provided is a method of screening a substance for an effect on a retinoid X receptor homodimer&#39;s ability to bind DNA comprising combining the substance with the homodimer and determining the effect of the compound on the homodimer&#39;s ability to bind DNA. A method of inhibiting an activity of a retinoid X receptor heterodimer comprising increasing the formation of a retinoid X receptor homodimer, thereby preventing the retinoid X receptor from forming a heterodimer and preventing the resulting heterodimer activity is also provided. A method of inhibiting an activity of a retinoid X receptor homodimer is also provided. A method of determining an increased probability of a pathology associated with retinoid X receptor homodimer formation and treating such pathology are further provided. Finally a method of screening a response element for binding to a retinoid X receptor homodimer is provided.

This invention was made with government support under Grant NumbersCA51993 and CA50676 from the National Institutes of Health. The U.S.Government may have certain rights in this invention.

This application is a continuation, of application Ser. No. 07/901,719,filed Jun. 16, 1992.

BACKGROUND OF THE INVENTION

The vitamin A metabolite all-trans-retinoic acid (RA) and its naturaland synthetic derivatives (retinoids) exert a broad range of biologicaleffects¹,2. Clinically, retinoids are important therapeutics in thetreatment of skin diseases and cancers³⁻⁶. Understanding how themultitude of retinoid actions can be mediated at the molecular level hasbeen greatly enhanced by the cloning and characterization of specificnuclear receptors, the retinoic acid receptors (RARs)⁷⁻¹² and theretinoid X receptors (RXRs)¹³⁻¹⁷. RARs and RXRs are part of thesteroid/thyroid hormone receptor superfamily¹⁸,19. Both types ofreceptors are encoded by three distinct genes, α, β, and γ, from which,in the case of RARs, multiple isoforms can be generated²⁰⁻²².Interestingly, while RARs are specific to vertebrates, the RXRs havebeen well conserved from Drosophila to man¹⁷,23. Despite theconsiderable advances in the understanding of the molecular mechanismsof retinoid receptor action in recent years, a central question ofwhether distinct molecular pathways for naturally occurring retinoidsexist has not yet been answered. The recent observation that the RAstereoisomer 9-cis-RA binds with high affinity to RXRs²³,24 suggested aretinoid response pathway distinct from that of all-trans-RA. However,it was almost simultaneously discovered by several laboratories thatRARs require interaction with auxiliary receptors for effective DNAbinding and function and that RXRs are such auxiliaryreceptors¹⁵,16,26-29. Hence, RARs appear to function effectively only asheterodimeric RAR/RXR complexes, or in combination with comparableauxiliary proteins that still need to be identified. Similarly, RXRswere shown to require RARs, thyroid hormone receptors (TRs), or VitaminD₃ receptors (VDRs) for effective DNA binding¹⁵,16,26-29. Thus, fromthese DNA binding studies, RXRs appeared to be able to functionpredominantly if not exclusively as auxiliary receptors, thereby playinga crucial role in generating a high degree of diversity and specificityof transcriptional controls and mediating the highly pleiotropic effectsof different hormones by increasing DNA affinity and specificity for atleast 3 different classes of ligand activated receptors.

Contrary to these findings, the present invention provides that RXRsform homodimers. The invention provides that these homodimerseffectively bind to specific response elements in the absence ofauxiliary receptors and their DNA binding specificity is distinct fromthat of the RXR containing heterodimers. The invention demonstrates anovel mechanism for retinoid action by which a ligand induced homodimermediates a distinct retinoid response pathway.

SUMMARY OF THE INVENTION

The invention provides a method of screening a substance for the abilityto affect the formation of a retinoid X receptor homodimer comprisingcombining the substance and a solution containing retinoid X receptorsand determining the presence of homodimer formation. Also provided is amethod of screening a substance for an effect on a retinoid X receptorhomodimer's ability to bind DNA comprising combining the substance withthe homodimer and determining the effect of the compound on thehomodimer's ability to bind DNA. A method of inhibiting an activity of aretinoid X receptor heterodimer comprising increasing the formation of aretinoid X receptor homodimer, thereby preventing the retinoid Xreceptor from forming a heterodimer and preventing the resultingheterodimer activity is also provided. A method of inhibiting anactivity of a retinoid X receptor homodimer is also provided. A methodof determining an increased probability of a pathology associated withretinoid X receptor homodimer formation and treating such pathology arefurther provided. Finally, a method of screening a response element forbinding with a retinoid X receptor homodimer is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows 9-cis retinoic acid induces RXR homodimer binding onTREpal.

(a) In vitro synthesized RXRα was incubated either with (+) or without(-) indicated hormones (10⁻⁷ M 9-cis-RA; 10⁻⁶ M RA; 10⁻⁶ M T₃) in thepresence or absence of in vitro synthesized TRα or RARβ for 30 min atroom temperature. After this preincubation, the reaction mixtures wereanalyzed by gel retardation assay using ³² P-labeled TREpal as probe.Lane 1 represents the nonspecific binding of unprogrammed reticulocytelysate. Open triangles indicate the nonspecific complex observed withunprogrammed reticulocyte lysate. Solid triangles indicate the specificTRα-RXRα heterodimer binding. Arrows indicate specific RXRα a homodimerbinding. The RXRα/RARβ heterodimer migrates at the same position as theRXRα homodimer. For comparison, the effect of 9-cis-RA on RARβ bindingis shown.

(b) To determine that 9-cis-RA induced DNA binding complex contains RXRαprotein, Flag-RXRα (F-RXR), an RXRα derivative that contains aneight-amino-acid epitope (Flag) at its amino terminal end which can berecognized by a specific anti-Flag monoclonal antibody, was constructed.In vitro synthesized F-RXRα was incubated either with (+) or without (-)10⁻⁷ M 9-cis-RA in the presence of either specific anti-Flag antibody(αF) or nonspecific preimmune serum (NI) for 30 min at room temperature.The effect of anti-Flag antibody on F-RXRα binding in the presence of9-cis-RA was then analyzed by gel retardation assay using ³² P-labeledTREpal as a probe. Lane 1 represents the nonspecific binding ofunprogrammed reticulocyte lysate (open triangles). Arrows indicates thespecific F-RXRα homodimer and RAR-RXR heterodimer binding. Diamondindicates the anti-Flag antibody up-shifted F-RXR homodimer.

FIG. 2 shows the characterization of 9-cis-RA induced RXR homodimer onTREpal.

(a) Cooperative binding of 9-cis-RA induced RXRα homodimer. Formation ofRXR-DNA complex at different receptor concentrations in the absence orpresence of 10⁻⁷ M 9-cis-RA was analyzed by gel retardation assay usinglabeled TREpal as probe. Open triangle indicates the nonspecific bindingof an unprogrammed reticulocyte lysate. Arrows indicate the specific RXRbinding complex.

(b) Quantitation of the RXR binding complex at different receptorconcentrations in the presence of 9-cis-RA. Gel slices containing RXRbinding complex in the presence of 9-cis-RA shown in FIG. 2(a) wereexcised and counted in a scintillation counter and plotted.

(c) 9-cis-RA concentration dependent binding of RXR homodimer on TREpal.Equal amounts of in vitro synthesized RXR protein were incubated withindicated concentrations of 9-cis-RA. The reaction mixtures were thenanalyzed by gel retardation assay using labeled TREpal as a probe. Opentriangles indicate the nonspecific binding of unprogrammed reticulocytelysate. Arrows indicate the specific RXR binding complex.

FIG. 3 shows 9-cis-RA induces RXR homodimer binding on RXR specificresponse elements.

(a) Nuclear receptor binding elements used in this study. Theseoligonucleotides were synthesized with appropriate restriction sites atboth ends as indicated by the small letters. Sequences that are closelyrelated to the AGG/TTCA motif are indicated by arrows.

(b) The effect of 9-cis-RA on RXR homodimer binding of ApoAI-RARE orCRBPII-RARE was analyzed essentially as described in FIG. 1a. Lane 1represents the nonspecific binding of unprogrammed reticulocyte lysatewhich are indicated by the open triangles. Solid triangles indicate theRAR/RXR heterodimer complex. Arrows indicate the specific RXR bindingcomplex.

FIG. 4 shows response element specific binding of RXR homodimer. Theeffect of 9-cis-RA on RXR binding on RA specific response elements (a),T₃ specific response elements (b), or estrogen specific response element(c) was analyzed by gel retardation assays as described in FIG. 1a. Forcomparison, the binding of RXR/RAR heterodimer (a), RXR/TR heterodimer(b) or estrogen receptor (c) is shown. Open triangles indicate thenonspecific binding of unprogrammed reticulocyte lysate. Solid triangleindicates the RAR/RXR heterodimer complex (a), TR/RXR heterodimercomplex (b) or ER complexes (c).

FIG. 5 shows RXR homodimerization occurs in solution. ³⁵ S-labeled invitro synthesized RXRα proteins was incubated with partially purifiedbacterially expressed Flag-RXR (F-RXR) (+) or similarly preparedglutathione transferase control protein (-) either in the presence orabsence of response elements or chemical cross-linker DSP as indicated.After incubation, either anti-Flag antibody (F) or nonspecific preimmuneserum (NI) was added. 10⁻⁷ M 9-cis-RA was maintained during workingprocess. The immune complexes were washed in the presence of 10⁻⁷M-cis-RA, boiled in SDS sample buffer and separated on a 10% SDS-PAGE.The ³⁵ S-labeled in vitro synthesized RXRα protein is shown in the rightpanel.

FIG. 6 shows transcriptional activation of RXR and RARα: RXRheterodimers by 9-cis RA on natural response elements. CV-1 cells werecontransfected with 100 ng of the reporter plasmids (a) TREpal-tk-CAT(b) βRARE-tk-CAT (c) ApoAI-RARE-tk-CAT and (d) CRBPI-RARE-tk-CAT and 5ng of empty pECE expression vector, pECE-RXRα, pECE RARα or combinationof both as indicated. Transfected cells were treated with no hormone(open bars), 10⁻⁷ M RA (shadowed bars) or 10⁻⁷ M 9-cis-RA (dark shadowedbars). The results of a representative experiment performed in duplicateare shown.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of screening a substance for the abilityto affect the formation of an RXR homodimer comprising combining thesubstance and a solution containing RXRs and determining the presence ofa homodimer formation. The presence of homodimer formation can, forexample, be determined by detecting the activation of transcription bythe RXR homodimer or by coprecipitation. The affect can be the inductionof homodimer formation, for example, an activity similar to thatactivated by 9-cis-RA. The affect can also be the inhibition ofhomodimer formation. Examples of inhibition include a substance whichbinds 9-cis-RA to block its activity. Such screening of substances isroutinely carried out given the subject discovery of homodimerformation. In particular assays set forth below can generally be usedfor screening by merely substituting the substance of interest for9-cis-RA. A good starting point for screening such "substances" is theactivity of 9-cis-RA described herein. The constituents on 9-cis-RA canbe varied and screened in the method to determine any increase ordecrease in homodimer formation. However, any substance can be screenedin this assay to determine any affect on homodimer formation.

The data set forth herein utilizes RXR. However, given the high homologybetween RXRα, β and γ, each protein should form homodimers and have theactivity described for RXRα homodimers. Relatedly, homodimers can formbetween different RXRs. For example, homodimers can form between RXRαand RXRβ or between RXRβ and RXRγ or between RXRα and RXRγ. The activityof these homodimers can be confirmed using the methods set forth herein.

The invention also provides a method of screening a substance for aneffect on an RXR homodimer's ability to bind DNA comprising combiningthe substance with the homodimer and determining the effect of thecompound on the homodimer's ability to bind DNA. For example, compoundswhich might bind the homodimer or bind the DNA response elementrecognized by an RXR homodimer can be screened in this method.

The invention further provides a method of inhibiting an activity of anRXR-containing heterodimer comprising increasing the formation of an RXRhomodimer, thereby preventing the RXR from forming a heterodimer andpreventing the resulting heterodimer activity. The activity can be anyactivity but is generally the activation or repression of transcription.The activity can be blocked, for example, by utilizing RXRs to formhomodimers which otherwise would be available to form heterodimers.Since the number of heterodimers are decreased, the activity of theheterodimers is decreased. In one example, the RXR heterodimer iscomprised of thyroid hormone receptor and RXR. The activity of theRXR/TR heterodimer was decreased. Other heterodimers can be tested usingstandard methods.

The invention also provides a method of inhibiting an activity of an RXRreceptor homodimer comprising preventing the formation of the RXRhomodimer. Such inhibition can be obtained, for example, by inhibiting9-cis-RA or the transcription of 9-cis-RA. The activity inhibited isgenerally the activation or repression of transcription.

The invention also provides a method of inhibiting an activity of an RXRhomodimer comprising preventing the binding of the RXR homodimer to itsresponse element. For example, the activity of a receptor which competesfor the same response element can be promoted. In general, the activitywhich is inhibited is the activation or repression of transcription.

The invention still further provides a method of determining anincreased probability of a pathology associated with RXR homodimerformation comprising detecting a decrease of RXR homodimer formation inthe subject when compared to a normal subject. Such a decrease canresult, for example, by a mutated RXR. The decrease can be detected byan assay for the homodimers in a sample or by detecting mutations knownto decrease homodimer formation.

Relatedly, the invention provides a method of treating a pathologyassociated with decreased RXR homodimer formation in a subjectcomprising increasing the amount of RXR capable of forming a homodimerin the subject. Such an increase can be accomplished, for example,utilizing compounds which promote RXR transcription. The pathology canbe associated with the skin, e.g., acne and psoriasis.

The invention also provides a purified RXR homodimer. By "purified" ismeant free of at least some of the cellular contaminants associated withRXR homodimers in a natural environment.

Finally, the invention provides a method of screening a response elementfor binding with a RXR homodimer comprising combining the responseelement with the RXR homodimer and detecting the presence of binding.The presence of binding can be determined by a number of standardmethods. In one method, binding is detected by the transcriptionalactivation of a marker which is operably linked to the response element.By "operably linked" is meant the marker can be transcribed in thepresence of the transcriptional activator.

The invention grows out of our study of the effects of the naturalvitamin A derivative 9-cis-RA on retinoid receptor DNA binding andtranscriptional activation. In contrast to all-trans-RA, the 9-cisderivative dramatically enhances RXRα binding at 10⁻⁹ to 10⁻⁸ Mconcentrations to several RXR specific RAREs but not to natural TREs orthe ERE. The effect is specific to RXR since 9-cis-RA did not inducebinding of RARα, β or γ to response elements (FIG. 1, FIG. 3). Judgingfrom the migration pattern in the gel shift assays, we assume that9-cis-RA induces homodimer formation, although a larger complexcontaining an RXR trimer or tetramer can occur (particularly in the caseof the CRBPII-RARE). Such trimer or tetramer formation can be testedusing the methods set forth herein.

RXRα homodimers exert response element specificity distinct fromheterodimers. The rCRBPI response element did not interact with RXRhomodimers, while the CRBPII response element 0 the only natural RAREidentified so far that contains perfect repeats--was a strong binder of9-cis-RA induced RXRα homodimers. It has been shown previously that thisresponse element is well activated by RXRα²⁴,30. Although this responseelement is also bound effectively by the RXRα-RARα heterodimer (FIG.3)²⁷,29, the heterodimer appears to have a repressor function³⁰.

The results obtained with the transcriptional activation studies agreewell with the DNA binding studies although CV-1 cells like all othermammalian tissue culture cells tested, contain endogenous retinoidreceptors that can partially obscure effects. Nonetheless, responseelements that strongly bound 9-cis-RA-RXRα homodimers also respondedstrongly to contransfected RXRα in the presence of 9-cis-RA whereasresponse elements that did not bind well to RXRα homodimers like therCRBPI-RARE or the MHC-TRE could not be activated by RXRα alone.

It is generally believed that the dimerization--homodimerization orheterodimerization--of nuclear hormone receptors is critical for highaffinity interaction of the receptor with their cognate responseelements. RXRs exist mainly as monomer in solution¹⁶ and require highconcentrations or the presence of RARs, TRs or VDR to display effectiveDNA binding activity¹³,15,16,26-30. The observation of the enhancedcooperative RXR DNA binding activity in the presence of 9-cis-RAdemonstrates that 9-cis-RA induced the formation of RXR homodimers whichhave an increased affinity for DNA. Thus, binding of 9-cis-RA to RXR caninduce a conformational change, which allows homodimerization to occur.It is interesting that although 9-cis-RA and RA can bind to RAR,²⁴,25they do not induce RAR homodimer formation.

RXRα homodimer formation can occur in solution in the absence of DNA.Thus, when 9-cis becomes available to cells, the equilibrium betweenmonomeric and dimeric receptors is changed and an additional species,the RXR homodimer can be formed, allowing for novel response pathways.The concept of ligand induced homodimer binding as observed by in vitrogel shift assay has not been previously observed for nuclear receptorswith the exception of a mutated estrogen receptor (ER-val-400)⁴³,44.

For the related TRs, a ligand effect has been reported on homodimerbinding, ⁴⁵,46 however the ligand (T3) reduced homodimer responseelement interaction. Since the carboxyterminal half of TRs and RARsencodes both ligand as well as dimerization functions³⁵,45,47, a strongeffect of the ligand on dimerization as observed here is not completelysurprising. However, the specificity of the effect is quite dramaticsince only homodimer but not heterodimer formation appears to beaffected. An overall picture emerges where the carboxyterminal region ofreceptors through its intermixed domains (that also includes atranscriptional activation region)⁴⁸ allows for multiple activities ofindividual receptors that may also include interactions with otherregulatory proteins⁴⁹.

The data presented in this application clearly demonstrate the centralrole of the RXRs, having dual functions that allow them to act asauxiliary receptors for three classes of hormone receptors, the RARs,TRs and VDRs through heterodimerization. The two functions ofRXR--homodimerization and heterodimerization--represent two distincttranscriptional regulatory controls that can be expected to affectdistinct physiological processes. Thus, 9-cis-RA can have therapeuticproperties distinct from that of all-trans RA.

9-cis induces RXR homodimer binding on the TREpal

Although RXRs have been shown to bind RA response elements (RAREs) whenused at high concentrations,²⁸,30,31 more recent investigations revealedthat RXR exists mainly as monomers in solution¹⁰ and that effective DNAinteraction requires heterodimer formation with RARs or TRs orVDR¹⁵,16,26-29. Binding of the heterodimers to a variety of responseelements was found to be ligand independent³². The newly discoverednatural RA isomer, 9-cis-RA, has been reported to be an effectiveactivator of RXRs in Drosophila Schneider cells that are known tocontain neither RAR nor TRs¹⁷,24. If 9-cis-RA is indeed a true ligandfor RXRs, one might expect that this ligand modulates RXR responseelement interaction. We therefore investigated the effect of 9-cis-RA onRXRα binding to the palindromic TRE (TREpal), an RXR responsiveelement¹⁴, in the absence and presence of coreceptors (TRs and RARs).

The cloning of the receptor cDNAs for RXRα, RARβ, or TRα into thepBluescript (Stratagene, San Diego, Calif.) have been describedpreviously ²⁶. Flag-RXRα was constructed as described previously³³ byligation of a double-stranded oligonucleotide containing an ATG codonand a DNA sequence encoding Flag (Arg-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) SEQID NO1! to the N-terminus of RXRα. The fusion product was then clonedinto pBluescript. The synthesis of receptor proteins using in vitrotranscription/translation system and gel retardation assays usingsynthesized receptor proteins and the double-stranded TREpal³⁴ were asdescribed³³. 9-cis-RA (m.p. 184°-187° C.) was prepared from9-cis-retinal by a two-step sequence of MnO₂ oxidation in the presenceof HOAc-MeOH to give the methyl ester (69%) followed by hydrolysis (80%)in 0.5N KOH in 25% aq. MeOH and crystallization (MeOH); HPLC (NovapakC₁₈, 32% MeCN, 27% MeCH, 16% APrOH, 24% H₂ O, 1% HOAc, 1.9 mL/min, 260nM) t_(R) 15.4 min (100%). To analyze the effect of hormones, thereceptor proteins were incubated with appropriate concentrations ofhormone at room temperature for 30 min before performing the DNA bindingassay. When anti-Flag antibody (Immunex, Seattle, Wash.) was used, 1 μlof the antiserum was incubated with receptor protein for another 30 minat room temperature before performing the DNA binding assay.

While in the absence of ligand RXR alone did not bind effectively to theTREpal and required TR or RAR for response element interaction, bindingof RXR was dramatically increased in the presence of 9-cis-RA (FIG. 1a)and did not require TRs or RARs. The RXR specific band observed in thepresence of 9-cis-RA was as prominent as the bands obtained with theheterodimeric TR-RXR and RAR-RXR complexes. The RXR complex migratedmore slowly than the TR-RXR complex at a position very similar to thatof the RAR-RXR heterodimer-TREpal complex. These data thereforedemonstrate that 9-cis-RA induces RXR homodimer formation. Due tomigration of the RAR-RXR complex at the same position as the 9-cis-RAinduced RXR homodimer complex, we were unable to determine in thisexperiment whether both complexes were formed. Remarkably, all-trans-RAwhen added at a concentration of 10⁻⁶ M, also induced to some degreeRXRα homodimer binding whereas T₃ did not. Although the RA effect couldbe observed with several freshly prepared RA solutions, it is not clearat this point whether this homodimer formation in the presence of RA isdue to 9-cis-RA impurities in the all-trans-RA solutions used here, oris a direct effect of all-trans-RA (see below). Interestingly, althoughit was reported that 9-cis-RA is capable of binding to RAR²⁴, unlike itseffect on RXR, 9-cis-RA was not found to induce RAR homodimer binding tothe TREpal.

To provide further evidence that the observed complex in the presence of9-cis-RA indeed was an RXR complex, we performed the DNA bindingexperiment with Flag-RXR, a derivative that carries an 8 amino acidaminoterminal epitope recognized specifically by anti-Flag (αF)antibody³³. As shown in FIG. 1b, αF supershifted the 9-cis-RA inducedcomplexes, while a nonspecific antibody did not. This proves that thecomplex observed with the TREpal in the presence of 9-cis-RA indeedcontained RXR protein. To examine how dependent the 9-cis-RA inducedhomodimer formation is on the concentration of RXR protein, increasingconcentrations of in vitro translated RXR protein were mixed with thelabeled TREpal in the presence or absence of 9-cis-RA (FIG. 2a). Whenthese mixtures were analyzed by the gel retardation assay, a strongcooperative effect in homodimeric DNA binding was seen, positivelydependent on the RXRα protein concentration (FIG. 2a,b). Although slightbinding of RXR can be observed when high concentrations of RXR wereused, 9-cis-RA was required for efficient complex formation at allreceptor concentrations used. We further determined the concentrationsof 9-cis-RA required for homodimer complex formation at all receptorconcentrations used. We further determined the concentrations of9-cis-RA required for homodimer complex formation and observed asignificant effect already at 10⁻⁹ M while optimal binding was seen at10⁻⁸ M (FIG. 2c). Thus, effective RXR homodimer DNA interaction isdependent on RXR protein concentration and can occur at low levels of9-cis-RA.

9-cis induced homodimeric interaction with specific response elementsRXR containing heterodimers have a highly specific interaction withvarious natural response elements in that TR-RXR heterodimers only bindstrongly to TREs but not to RAREs, whereas the opposite is true forRAR-RXR heterodimers¹⁵,32. We examined the sequence requirement of DNAbinding of 9-cis-RA induced RXR homodimer using a number of natural andsynthetic response elements (FIG. 3a).

Gel retardation assays using in vitro synthesized receptor protein aredescribed in the FIG. 1 legend. The following oligonucleotides and theircomplements were used as probes in FIGS. 3 and 4. ApoAI-RARE, a directrepeat response element with 2 bp spacer³¹,gatcAGGCAGGGGTCAAGGGTTCAGTgatc SEQ ID NO2!; CRBPII-RARE, a direct repeatRXR specific response element with 1 bp spacer³⁰,gatcCAGGTCACAGGTCACAGGTCACAGTTCAAgatc SEQ ID NO3!; βRARE, a directrepeat of RA response element present in RARFβ promoter³⁵,36,gatcTGATAGGGTTCACCGAAAGTTCACTCagatc SEQ ID NO4!; CRBPI-RARE, a directrepeat RA specific response element present in rat CRBPI promoter³⁷,gatccAGGTCAAAAAAGTCAGgatc SEQ ID NO5!; MHC-TRE, a direct repeat T₃specific response element present in rat α-myosin heavy chain gene³⁹,gatcCTGGAGGTGACAGGAGGACAGCgatc SEQ ID NO6!; ME-TRE, a direct repeat T₃specific response element present in the rat malic enzyme gene⁴⁰,gatcCAGGACGTTGGGGTTAGGGGAGGACAGTGGgatc SEQ ID NO7!; DR-4, an idealizeddirect repeat T₃ specific response element with 4 bp spacer³⁶,gatcTCAGGTCATCTCAGGTCAgatc SEQ ID NO8!; DR-5, an idealized direct repeatRA specific response element with 5 bp spacer³⁸,gatcTCAGGTCATCCTCAGGTCAgatc SEQ ID NO9!; ERE, a perfect pal indromic ERresponse element⁴¹, gatcTCAGGTCACTGTGACCTGAgatc SEQ ID NO10!. Thesequence of TREpal SEQ ID NO 11! is shown for comparison.

ApoAI-RARE (a direct repeat response element that contains a 2 bpspacer) which has been suggested to be RXR specific³¹ resulted in astrong RXR complex in the presence of 9-cis-RA and to a lesser degreewith RA (10⁻⁶ M). The RXR-RAR heterodimer also bound effectively to thisresponse element. Since the heterodimer complex migrated at the sameposition as RXR homodimers, the effect of 9-cis-RA on RXR-RARheterodimers cannot be clearly determined. RAR homodimer binding was notinduced by 9-cis-RA (FIG. 3b). When we investigated 9-cis-RA induced RXRbinding to another RXR responsive element³⁰, the CRBPII-RARE, weobserved a complex that migrated more slowly than the heterodimer (inthe absence of ligand), while in the presence of 9-cis-RA and RAR, bothhomodimer and heterodimer binding appeared to be reduced in theirintensity (FIG. 3); a similar effect was seen at high RA concentrationsor when both 9-cis-RA and RA were present. 9-cis-RA also induced RXRinteraction with the β RARE (FIG. 4a), the RAR response element from thehuman RARβ promoter³⁵,36 that contains a 5 bp spacer. However, the RXRhomodimer band was considerably weaker than the RAR-RXR heterodimer bandat the protein concentrations used. Interestingly, another natural RAREderived from the rat CRBPI promoter³⁷, that similar to the ApoAI-RAREcontains a 2 bp spacer did not show any binding of RXR in the presenceof 9-cis-RA (FIG. 4a), indicating that the actual sequence of therepeated core motif is critical for RXR homodimer binding. Similarly,the DR-5-RARE, a perfect repeat element derived from the β-RARE³⁸ didnot exhibit interaction with RXR in the presence of 9-cis-RA, while itinteracted strongly with RXR when RARE was present (FIG. 4a).

To examine whether RXR homodimer binding is specific to certain RAREs,we also performed gel shift experiments with the T₃ response elementsfrom the rat α-myosin heavy chain promoter (MHC-TRE)³⁹, the rat malicenzyme (ME-TRE)⁴⁰ and the perfect repeat DR-4³⁸. In all three cases,specific binding of RXR in the presence or absence of 9-cis-RA could notbe observed, while all three response elements bound effectively TR/RXRheterodimers (FIG. 4b), consistent with the notion that these elementsare not induced by retinoids. Similarly, the perfect palindromic ERE⁴¹also did not interact with RXR homodimers (FIG. 4c).

9-cis induced homodimer formation occurs in the absence of DNA

It was reported the RXR exists mainly as monomer in solution¹⁶. Animportant question is whether 9-cis-RA induced RXR homodimers, like theRXR containing heterodimers, can form in solution in the absence of DNA.To address this question we took advantage of the Flag-RXR derivativethat can be specifically precipitated with anti-Flag antibody while RXRwild type cannot.

Flag-RXRα was cloned in frame in the expression vector pGex 2T(Pharmacia) and was expressed in bacteria using the procedure providedby the manufacturer. Protein was partially purified on a prepackedglutathione sepharose 4B column (Pharmacia) and tested for its functionby gel retardation assays and western blotting using anti-Flag antibody.Immunocoprecipitation assay was performed essentially as described²⁶.Briefly, 10 μl of ³⁵ S-labeled in vitro synthesized RXRα protein wasincubated with 5 μl (approximately 0.1 μg) of partially purifiedbacterially expressed Flag-RXRα fusion protein or similarly preparedglutathione transferase control protein in 100 μl buffer containing 10⁻⁷M 9-cis-RA, 50 mM KCl and 10% glycerol for 30 min at room temperature.When assayed in the presence of chemical cross-linker oroligonucleotides, we added 2 μl of 100 mM Dithiobissuccinimidylpropionate (DSP) dissolved in DMSO or 10 ng ofoligonucleotide and continued the incubation at room temperature for 15min. The reaction mixtures were then incubated with 1 μl of anti-Flagantibody or nonspecific preimmune serum for 2 h on ice. Immune complexeswere precipitated by adding 50 μl of protein-A-sepharose slurry andmixing continuously in the cold room for 1 h. The immune complexes werewashed extensively with cold NET-N buffer (20 mM Tris, pH 8.0, 100 mMNaCl, 1 mM DTT, 0.5% NP-40) containing 10⁻⁷ M 9-cis-RA, boiled in SDSsample buffer and resolved by SDS-polyacrylamide gel electrophoresis.The gel was fixed, dried and visualized by autoradiography.

When we mixed Flag-RXR with in vitro labeled ³⁵ S-RXR protein, thelabeled RXR could be coprecipitated in the presence of anti-Flagantibody but not in the presence of nonspecific serum (FIG. 5).Coprecipitation efficiency was slightly increased in the presence of theApoAI-RARE but not in the presence of the MHC-TRE. In addition,incubation with a crosslinker (DSP) further enhanced coprecipitation ofthe labeled RXR. In all cases, specific coprecipitation was onlyobserved in the presence of 9-cis-RA. These data therefore give strongsupport to the assumption that 9-cis-RA induced RXR homodimer formationoccurs in solution and does not require RXR-DNA interaction.

Response element specific transcriptional activation by 9-cis-RA andRXRα

To investigate whether 9-cis-RA/RXRα homodimer response elementinteraction can be correlated with transcriptional activation of suchresponse elements by the 9-cis-RA/RXR complex, we carried out a seriesof transient transfection assays in CV-1 cells, where we cotransfectedreceptor expression vectors with CAT reporter constructs that carriedvarious response elements upstream of the tk promoter. CV-1 cells weretransiently transfected using a modified calcium phosphate precipitationprocedure as described previously⁴². CAT activity was normalized fortransfection efficiency by measuring the enzymatic activity derived fromthe cotransfected β-galactosidase expression plasmid (pCH110,Pharmacia). The transfected cells were grown in the absence or presenceof 10⁻⁷ M 9-cis-RA or all-trans-RA.

With the TREpal containing reporter, we observed strong activation byRXRα in the presence of 9-cis-RA and little activation when RA wasadded. Activation could be further enhanced by cotransfection of RARα.In this case, however, RA also functioned as an effective activatoralthough not as efficiently as 9-cis-RA. Overall, activation by 9-cis-RAin the presence of RARα and RXRα was approximately twice as strong asseen with RXRα alone, consistent with the DNA binding data where bindingwas observed by both the heterodimer and the homodimer in the presenceof 9-cis-RA when both receptors were present (FIG. 6a). When we examinedthe βRARE (FIG. 6b), we observed that this response element was highlyactivated by endogenous CV-1 cell receptors consistent with previousobservations³⁶,41, such that further activation by low concentrations ofcotransfected receptors could not be observed. Interestingly, however,9-cis-RA was a more potent activator at 10⁻⁷ M than RA. These data thusindicate that CV-1 cells contain endogenous retinoid receptor activitythat is particularly active on the βRARE and is responsive to 9-cis-RA.

The ApoAI element containing reporter was also very effectivelyactivated by RXRα in the presence of 9-cis-RA (FIG. 6c) while RA did notinduce above the level obtained in the absence of RXRα. Similar to theTREpal, maximal activation was seen when both receptors RXRα and RARαwere cotransfected. Under these conditions RA also led to a strongactivation. In contrast, the CRBPI element, where we did not observe DNAbinding by RXR in the presence of 9-cis-RA, also was not activated byRXR and 9-cis-RA in the transient transfection studies (FIG. 6d) whileRARα alone led to significant activation that was mostly 9-cis-RAdependent. The heterodimer RARαRXRα allowed maximal activation in thepresence of 9-cis-RA. Not unexpectedly, no induction by RXRα and9-cis-RA was observed on the MHC-TRE, the ME-TRE or the ERE. These invivo analyses showed a very significant correlation to the resultsobtained with the in vitro DNA binding studies, in that strongactivation by RXRα in the presence of 9-cis-RA is only observed on theresponse elements that strongly interact with the 9-cis-RA induced RXRαhomodimer.

9-cis retinoic acid inhibits activation by TR/RXR heterodimer

We have observed that a CAT reported gene that is activated by a thyroidhormone receptor/RXR heterodimer in the presence of thyroid hormone (T₃)can be inhibited by adding 9-cis-RA. This type of inhibition is mosteasily measured by using a transfection assay.

The preceding examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed.

Throughout this application various publications are referenced bynumbers. Following is a complete citation to the publications. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

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2. Roberts, A. B., & Sporn, M. B. In: The Retinoids (M. B. Sporn, A. B.Roberts, D. S. Goodman, eds). Academic Press, Florida pp. 209-286(1984).

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16. Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M.,Zacharewski, T., Chen, J-Y., Staub, A., Garnier, J-M., Mader, S., &Chambon, P. Cell 68, 377-395 (1992).

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgTyrLysAspAspAspAspLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GATCAGGCAGGGGTCAAGGGTTCAGTGATC30                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GATCCAGGTCACAGGTCACAGGTCACAGTTCAAGATC37                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GATCTGATAGGGTTCACCGAAAGTTCACTCAGATC35                                         (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GATCCAGGTCAAAAAAGTCAGGATC25                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GATCCTGGAGGTGACAGGAGGACAGCGATC30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GATCCAGGACGTTGGGGTTAGGGGAGGACAGTGGGATC38                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATCTCAGGTCATCTCAGGTCAGATC26                                                  (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GATCTCAGGTCATCCTCAGGTCAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GATCTCAGGTCACTGTGACCTGAGATC27                                                 (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GATCTCAGGTCATGACCTGAGATC24                                                    __________________________________________________________________________

What is claimed is:
 1. A method of screening a substance for the abilityto affect the formation of a functional retinoid X receptor homodimer,which affects the activation of transcription by the homodimercomprising combining the substance and a solution containing retinoid Xreceptors and determining the presence of homodimer formation insolution by either 1) a gel shift assay utilizing a response element tobind the homodimer or 2) coprecipitation, the presence of homodimerformation indicating a substance that can affect the activation oftranscription.
 2. The method of claim 1, wherein the effect is theinduction of homodimer formation.
 3. The method of claim 1, wherein theretinoid X receptor is retinoid X receptor α.
 4. The method of claim 1,wherein the presence of homodimer formation is detected by saidcoprecipitation and said coprecipitation is resolved by a denaturing gelelectrophoresis assay.
 5. The method of claim 1, wherein the presence ofhomodimer formation is detected by said gel shift assay and said gelshift assay is resolved under non-denaturing conditions.
 6. A method ofscreening a substance for the ability to inhibit the formation of aretinoid X receptor homodimer, which affects the activation oftranscription by the homodimer, comprising the steps of:a. contacting ina first solution, retinoid X receptors and a ligand under conditionsthat allow retinoid X receptor homodimer formation; b. determining afirst amount of retinoid X receptor homodimer formation in said firstsolution by a method selected from the group consisting of 1) a gelshift assay utilizing a response element to bind the homodimer and 2)coprecipitation; c. contacting in a second solution, retinoid Xreceptors, a ligand and a substance under said conditions of step a); d.determining a second amount of retinoid X receptor homodimer formationin said second solution by the method selected in step b); and e.thereafter comparing said first amount of retinoid X receptor homodimerformation and said second amount of retinoid X receptor homodimerformation, wherein a determination that said first amount is greaterthan said second amount indicates said substance inhibits the formationof a retinoid X receptor homodimer, the inhibition of homodimerformation indicating a substance that can affect the activation oftranscription.
 7. A method of screening a response element for bindingwith a retinoid X receptor homodimer, comprising combining in solutionthe response element with the retinoid X receptor homodimer complexedwith a ligand selected from the group consisting of 9-cis-RA andall-trans-RA, and detecting the presence of binding by a gel shiftassay.
 8. The method of claim 7, wherein said gel shift assay isresolved under non-denaturing conditions.